The insulin receptor (IR) serves to focus the hormone on particular target tissues as well as to initiate the response of these cells to the hormone via its intrinsic tyrosine kinase activity which is critical for various biological responses. A major question that still needs to be answered is how this IR kinase and subsequent signaling is regulated since patients with non-insulin dependent diabetes exhibit a reversible decrease in IR signalling, possibly contributing to their insulin resistance. One potential mechanism would be an increase in the ser/thr phosphorylation of the IR and/or subsequent molecules in the signaling cascade. During the prior grant period, extensive studies were focused on the regulation of IR signaling by the protein kinase C pathway. Activation of this pathway was shown to modulate insulin-stimulated signaling via an increase in the serine phosphorylation of a specific serine residue in one of the substrates (called IRS-1) of the insulin receptor kinase. In the next grant period we plan to further test the role of this residue in the ability of other agents to modulate IR signaling including via various counter regulatory hormones such as tumor necrosis factor and PDGF. In addition, we will attempt to identify various kinases capable of phosphorylating this residue including an insulin-stimulated kinase which appears to be able to readily phosphorylate this regulatory serine. Such serine phosphorylation of IRS-1 and other substrates of the IR tyrosine kinase may contribute to the insulin resistance observed in various models of NIDDM. After tyrosine phosphorylation, insulin-receptor substrates like IRS-1, 2 and 3 are bound and activate a lipid kinase called PI 3- kinase. This kinase produces phosphatidylinositol 3-phosphates which can activate various ser/thr kinases including one called Akt/PKB as well as certain isoforms of the protein kinase C family of kinases. During the last grant period we have shown that the enzymatic activity of Akt/PKB is greatly activated in insulin treated cells. In addition, we have shown that constitutively active forms of the enzyme can mediate a number of insulin-like biological responses including the stimulation of glucose uptake, GLUT4 translocation, activation of the 70 kDa S6 kinase and stimulation of mTOR enzymatic activity. In the present proposal we plan to further explore the mechanism whereby Akt can induce various biological responses. In particular, we plan to identify potential substrates of the Akt kinase as well as to investigate further the mechanism whereby Akt is capable of eliciting the above biological responses as well as several additional biological responses. Finally, we plan to explore the role of Akt in mediating the negative regulation of the IR signaling pathway.